The outlines of an SV40 DNA replication scheme are now in hand. The SV40 replicative intermediates have been isolated and can be employed to reconstruct a DNA replication apparatus in vitro. Techniques are described for the isolation and characterization of both viral and cellular proteins involved in DNA replication. SV40 T-antigen may be coded for by the viral tsA gene. T-antigen binds preferentially to some DNA's (like SV40) and not others (lambda, polyoma). A new DNA binding assay has been developed (Sepharose-DNA chromatography) that should permit the purification of T-antigen and an experimental test of whether T-antigen is coded for by the tsA gene. An assay for SV40 specific surface antigens has also been developed and the relationship between this surface antigen and T-antigen will be tested. Various intracellular forms of DNA polymerase, unwinding proteins, ligase, RNA polymerases, etc. will be employed with replicating SV40 DNA (as a template) in vitro to elucidate the functions required for viral DNA replication. SV40 can complement an adenovirus, DNA negative, ts mutant that codes for a DNA unwinding protein. SV40 infection supplies a functional unwinding protein and this will be isolated and characterized. The goal of these studies is to understand the biochemistry of viral and eukaryotic DNA replication, genetic recombination and transformation. With both SV40 and adenovirus those functions involved in DNA replication are also involved in viral transformation. Thus the study of DNA replication should be useful in understanding the role of viral functions in transformation.